The present invention relates to an oligonucleotide library, more particularly a chimaeric oligonucleotide library, and uses thereof in the identification of antisense binding sites in target mRNA and in providing potential therapeutic agents.
Antisense oligonucleotides are single-stranded nucleic acids which are complementary to the coding or xe2x80x9csensexe2x80x9d strand of genetic material. An antisense oligonucleotide is therefore also complementary to the mRNA produced from the genetic material. If antisense DNA or RNA is present in a cell with the mRNA, hybridisation takes place to form a duplex thereby preventing translation of the mRNA by ribosomes to make a protein. Thus, antisense RNA can be used to block the expression of genes that make proteins.
The antisense approach to the inhibition of gene expression, though conceptually straightforward, presents technologically demanding challenges. A variety of approaches have been taken by various academic groups and biotechnology companies. Oligonucleotides have been made with sugar modifications, such as 2xe2x80x2-O allyl ribonucleotides, and with backbone modifications in the phosphate group, such as phosphorothioate deoxyribonucleotides. However, production of these individual oligonucleotides for application as antisense therapeutics, reagents or tools for drug target validation has been hampered because methods of identifying potentially efficacious antisense compounds against a given target mRNA are extremely difficult. Even with an mRNA of known sequence, it is often impossible to predict what sub-sequences in the target mRNA might be available for antisense binding because of the three-dimensional structure of the mRNA and the association of RNA with proteins.
In an alternative approach to the use of chemically modified oligonucleotides, Lieber and Strauss (ref 22) report the use of a ribozyme expression library for the purpose of selecting cleavage sites in target RNAs. The ribozyme approach suffers from the disadvantage that it requires cleavage sites containing GUC or CUC and thus is not generally applicable to all possible cleavage sites. In addition, cleavage efficiency is relatively low, and chemical synthesis of ribozyme libraries is difficult.
According to one aspect, the present invention provides a chimaeric oligonucleotide library for use in identifying an antisense binding site in a target mRNA, which comprises a plurality of distinct chimaeric oligonucleotides capable of hybridizing to mRNA to form a duplex, the nucleotide sequences of which each have a common length of 7 to 20 bases and are generated randomly or generated from information characterising the sequence of the target mRNA, wherein substantially all the nucleotide sequences of said common length which are present as sub-sequences in the target mRNA are present in the library, and wherein each nucleotide sequence comprises:
a) a recognition region comprising a sequence of nucleotides which is recognisable by a duplex-cutting RNAase when hybridized to the mRNA, and
b) a flanking region comprising a sequence of chemically-modified nucleotides which binds to the mRNA sufficiently tightly to stabilise the duplex for cutting of the mRNA in the duplex by the duplex-cutting RNAase, wherein the nucleotides constituting the flanking region are different from those constituting the recognition region; and wherein each oligonucleotide is protected against exonuclease attack.
The mRNA may be from human or other mammalian origin or from invertebrates. The term mRNA as used herein also encompasses the corresponding RNA from plants, viruses and bacteria.
Chimaeric Oligonucleotide
Each chimaeric oligonucleotide forming the library may be made synthetically using any commonly-available oligonucleotide sequence synthesizer. The exact length of the nucleotide sequence will reflect a balance between achieving the necessary specificity and keeping the length to a minimum to minimise cost. Preferably, the nucleotide sequence has a length in the range 10 to 20, more preferably 14 to 17 bases, yet more preferably around 15 bases.
The oligonucleotide is preferably protected against-nuclease attack so as to minimise degradation in the cell and increase its stability. This is particularly important in the design of an antisense compound for therapeutic use. Protection against exonuclease attack may be achieved by protecting one or preferably both ends of the oligonucleotide, for example by reverse T. or any other well-known method. Selection of the nucleotides constituting the recognition and flanking regions may also contribute to stability against nuclease because some nucleotides are more nuclease-resistant than others.
Preferably, each chimaeric oligonucleotide comprises two flanking regions, one on either side of the recognition region. In this way, the recognition region may be thought of as a xe2x80x9cwindowxe2x80x9d flanked by the two flanking regions so as to form with the mRNA a substrate for the duplex-cutting RNAase. In a preferred embodiment, each of the flanking regions is protected against exonuclease attack, preferably by reverse T. A preferred duplex-cutting RNAase is RNAase H, advantageously endogenous RNAase H (Ref 23).
The nucleotides constituting the recognition region are either modified or unmodified nucleotides and are preferably deoxyribonucleotides or phosphorothioate deoxyribonucleotides (see FIG. 4c). These nucleotides are recognisable by RNAase H when hybridized to mRNA. Typically, the recognition region comprises at least four nucleotides, preferably 5 to 10 nucleotides. In a particularly preferred embodiment, the recognition region comprises five nucleotides.
The nucleotides constituting the flanking region are chemically modified so as to increase the binding constant of the oligonucleotide for hybridization to the target mRNA and preferably to increase stability of the oligonucleotide in vivo. For a particular antisense oligonucleotide, the efficiency of hybridization to mRNA is a function of concentration. Thus, to improve hybridization as a given concentration, the stability of the hybrid duplex must be increased. A number of chemical modifications can be introduced into the oligonucleotide for this purpose and these fall into three broad classes (see also FIG. 1, regions 1, 2 and 3):
Sugar Modifications
Various modifications to the 2xe2x80x2 position in the sugar moiety may be made. For example, both 2xe2x80x2-O methyl oligoribonucleotides and 2xe2x80x2-O allyl oligoribonucleotides may be useful (see references 1 and 2 and see also FIG. 2a and b). These analogues do not form hybrid duplexes with RNA which are substrates for RNAase H. In a particularly preferred embodiment of the present invention, two flanking regions, each having four or five of one of the modified sugar-containing oligoribonucleotides, flank a window region of four or five normal deoxyribonucleotides. The window region will thereby allow cleavage of the mRNA and the sugar-modified flanking regions increase the binding of the chimaeric oligonucleotide to the mRNA. Other 2xe2x80x2 sugar modifications which may be used include F-substituted and NH2-substituted oligoribonucleotides (see FIGS. 2c and 2d and references 3 and 4).
Base Modifications
The chemically-modified nucleotides constituting the flanking region may be modified in the base moiety. The propyne analogues of dT and dC, 5-propynyl deoxyuridine (see FIG. 3a) and 5-propynyl deoxycytidine (see FIG. 3b), both increase the duplex hybridization temperature and stabilize the duplex. This stabilization may be due to increased strength of hydrogen bonding to each Watson-Crick partner or increased base stacking (or both). 2-amino adenine is an analogue of dA (see FIG. 3c) and also increases the stability of the duplex. This may be due to the formation of a third hydrogen bond with thymine. The 2-amino adenine-thymine base pair is intermediate in stability between a G.C and a A.T base pair.
Phosphate Modifications
The chemically-modified nucleotides constituting the flanking region may be modified in the phosphate moiety. Under certain conditions such as low salt concentration, analogues such as methylphosphonates (FIG. 4a), triesters (FIG. 4b) and phosphoramidates (FIG. 4e) have been shown to increase duplex stability. The hybrid duplexes are not substrates for RNAase H. Further phosphate modifications include phosphorodithirates (FIG. 4d) and boranophosphates (FIG. 4f), each of which increase the stability of oligonucleotides.
Isosteric replacement of phosphorus by sulphur gives nuclease resistant oligonucleotides. (see reference 14). Replacement by carbon at either phosphorus or linking oxygen is also a further possibility (see FIG. 5).
In use, the chimaeric oligonucleotide of the invention acts as an antisense compound by specifically binding to target mRNA at an antisense binding site so that cleavage or cutting of the mRNA by a duplex-cutting RNAase takes place there. The chimaeric oligonucleotide will bind to the target mRNA to form a duplex. The recognition region is recognised by a duplex-cutting RNAase. The flanking region renders the duplex sufficiently stable to enable the RNAase to cut the mRNA in the duplex efficiently. Once cut, the mRNA and the oligonucleotide detach thereby leaving the oligonucleotide to bind a further mRNA. In this way, the chimaeric oligonucleotide acts catalytically.
Oligonucleotide Libraries
In a further aspect, the present invention provides use of an oligonucleotide library in a method of identifying an antisense binding site in a target mRNA, wherein the oligonucleotide library comprises a plurality of distinct nucleotide sequences, each having a common length in the range 7 to 20 bases, preferably 10 to 20 bases, and each of which comprises a substrate for a duplex-cutting RNAase if hybridised to the mRNA, which library is generated randomly, or generated from information characterising the sequence of the target mRNA, so that substantially all nucleotide sequences of said common length which are present as sub-sequences in the target mRNA are present in the library. The nucleotide sequences may comprise modified nucleotides, such as phosphorothioates, as described herein. The nucleotide sequences may be chimaeric or non-chimaeric.
In one embodiment, the library is generated randomly by means of an oligonucleotide sequence synthesizer. An aim of generating the sequences randomly is that substantially all possible nucleotide sequences of the specified length are generated. For a sequence of 10 bases in length (a 10-mer), 410 distinct nucleotide sequences would need to be generated to cover all possibilities. This works out as approximately 106 distinct nucleotide sequences. For a 15-mer, the library would need approximately 109 to 1010 nucleotide sequences. Each nucleotide sequence will have a common length (i.e. they will all be 10-mers or will all be 11-mers, etc.). Any commonly-available oligonucleotide sequence synthesizer may be used for this purpose such as supplied by Applied Biosystems. All four possible bases are fed into the machine with an appropriate program using suitable nucleotides or modified nucleotides.
In an alternative embodiment, instead of generating the nucleotide sequences randomly they are generated from information characterising the sequence of the target mRNA. The sequence of the target mRNA needs to be known and can then be programmed into the oligonucleotide sequence synthesizer. For example, in the case of a gene which produces an mRNA of 450 nucleotides, a library of 15-mers would be produced with a total of 436 distinct nucleotide sequences (i.e. length of mRNA minus length of nucleotide sequence plus 1). In this way, all potential sub-sequences of the mRNA would be represented in the library. This is advantageous over the random generation of the library because there is no dilution of potentially useful nucleotide sequences by randomly generated sequences not present in the target mRNA. A further way of ensuring that all sub-sequences of the mRNA are present in the library is to produce, in the case of an mRNA of 450 nucleotides, a library of 30 15-mers (i.e. length of mRNA divided by the length of the nucleotide sequence).
In a further aspect, the present invention provides a method of identifying an antisense binding site in a target mRNA, which comprises:
1) incubating with the target mRNA an oligonucleotide library and a duplex-cutting RNAase under conditions to produce target mRNA cut at the antisense binding site; and
2) identifying the antisense binding site from the position of the cut in the mRNA; wherein the oligonucleotide library comprises a plurality of distinct nucleotide sequences, each having a common length in the range 7 to 20 bases, preferably 10 to 20 bases, and each of which comprises a substrate for the duplex cutting RNAase if hybridized to the mRNA; and wherein the oligonucleotide library is generated randomly, or generated from information characterising the sequence of the target mRNA, so that substantially all nucleotide sequences of such common length which are present as sub-sequences in the target mRNA are present in the library.
Use of an oligonucleotide library in this manner enables identification of one or more antisense binding sites in a target mRNA and such identification can be achieved very rapidly in comparison with known methods. No information about the three-dimension structure of the mRNA is required because the identification of the antisense binding sites is empirical. Incubation of the target mRNA with the oligonucleotide library and the duplex-cutting RNAase can, by suitable variation of the reaction conditions, produce target mRNA cut at one or more antisense binding sites. This is because the library will contain one or more oligonucleotides which are complementary to such binding sites and will bind thereto under appropriate conditions to form a duplex. The duplex acts as a substrate for the duplex cutting RNAase. When the mRNA is cut at the binding site the oligonucleotide is released and is thereby made available for further binding. The oligonucleotide therefore acts catalytically and the duplex-cutting RNAase acts enzymatically. The duplex-cutting RNAase is separate from the oligonucleotide library and is preferably from a cell extract. Preferably, the duplex-cutting RNAase is RNAase H. The target mRNA is also preferably from a cell extract. Advantageously, therefore, both RNAase H and mRNA are present in the same cell extract with which the oligonucleotide library is incubated.
The position of the cut in the mRNA may be determined by sequencing isolated cut target mRNA. Preferably, the cut target mRNA is amplified prior to isolation, for example by reverse transcription and polymerase chain reaction.
Once the antisense binding site is identified from the position of the cut in the mRNA and sequenced, an antisense oligonucleotide may be synthesized which is capable of binding to the site. In this way, a method is provided for the production of an antisense oligonucleotide. Thus, a chimaeric oligonucleotide of the type discussed above can be obtained. An important use of such a chimaeric oligonucleotide is as a therapeutic agent capable of hybridising to a specific antisense binding site in a target mRNA. The nucleotide sequence of the chimaeric oligonucleotide needs to be specific to the antisense binding site for this purpose.
In summary, using the library approach described herein, optimal sequences of effective antisense compounds can be identified against specific mRNA targets. The antisense compounds are useful as potential therapeutics, as tools for drug target validation, in diagnostics and as a research reagent.